The environment in which humans live is replete with pest infestation. Pests including insects, arachnids, crustaceans, fungi, bacteria, viruses, nematodes, flatworms, roundworms, pinworms, hookworms, tapeworms, trypanosomes, schistosomes, botflies, fleas, ticks, mites, and lice and the like are pervasive in the human environment, and a multitude of means have been utilized for attempting to control infestations by these pests. Compositions for controlling infestations by microscopic pests such as bacteria, fungi, and viruses have been provided in the form of antibiotic compositions, antiviral compositions, and antifungal compositions. Compositions for controlling infestations by larger pests such as nematodes, flatworm, roundworms, pinworms, heartworms, tapeworms, trypanosomes, schistosomes, and the like have typically been in the form of chemical compositions which can either be applied to the surfaces of substrates on which pests are known to infest, or to be ingested by an infested animal in the form of pellets, powders, tablets, pastes, or capsules and the like. The present invention is directed to providing an improved means for controlling pest infestation compared to the compositions known in the art.
Commercial crops are often the targets of insect attack. Substantial progress has been made in the last a few decades towards developing more efficient methods and compositions for controlling insect infestations in plants. Chemical pesticides have been very effective in eradicating pest infestations. However, there are several disadvantages to using chemical pesticidal agents. Chemical pesticidal agents are not selective. Applications of chemical pesticides are intended to control invertebrate pests that are harmful to various crops and other plants. However, because of the lack of selectivity, the chemical pesticidal agents exert their effects on non-target fauna as well, often effectively sterilizing a field for a period of time over which the pesticidal agents have been applied. Chemical pesticidal agents persist in the environment and generally are slow to be metabolized, if at all. They accumulate in the food chain, and particularly in the higher predator species. Accumulations of these chemical pesticidal agents results in the development of resistance to the agents and in species higher up the evolutionary ladder, act as mutagens and/or carcinogens often causing irreversible and deleterious genetic modifications. Thus there has been a long felt need for environmentally friendly methods for controlling or eradicating insect infestation on or in plants, i.e., methods which are selective, environmentally inert, non-persistent, and biodegradable, and that fit well into pest resistance management schemes.
Compositions that include Bacillus thuringiensis (B.t.) bacteria have been commercially available and used as environmentally safe and acceptable insecticides for more than thirty years. The insecticidal effect of Bt bacteria arises as a result of proteins that are produced exclusively by these bacteria that do not persist in the environment, that are highly selective as to the target species affected, exert their effects only upon ingestion by a target pest, and have been shown to be harmless to plants and other non-targeted organisms, including humans. Transgenic plants containing one or more genes encoding insecticidal B.t. protein are also available in the art and are remarkably efficient in controlling insect pest infestation. A substantial result of the use of recombinant plants expressing Bt insecticidal proteins is a marked decrease in the amount of chemical pesticidal agents that are applied to the environment to control pest infestation in crop fields in areas in which such transgenic crops are used. The decrease in application of chemical pesticidal agents has resulted in cleaner soils and cleaner waters running off of the soils into the surrounding streams, rivers, ponds and lakes. In addition to these environmental benefits, there has been a noticeable increase in the numbers of beneficial insects in crop fields in which transgenic insect resistant crops are grown because of the decrease in the use of chemical insecticidal agents.
The adventitious use of recombinant plants expressing an insecticidal crystal protein toxin has strengthened the concern about the spontaneous development of resistance to the toxin in the target pest population. One means for delaying or eliminating the onset of resistance in the pest population is to combine the recombinant toxin with a second means for controlling the pests in which the second means exerts its effects through a different mode of action in comparison to the recombinant toxin. One means for deploying two or more toxins would be to incorporate a seed treatment containing some insecticidal composition that is effective at very low doses and which infuses into the soil or into the growing recombinant plant after sprouting. This means has been shown to be effective and economical, but has the disadvantage of subjecting the environment to a chemical pesticide that may accumulate in the food chain and that may persist in the environment. Another means would be to deploy a recombinant plant that expresses at least two different insecticidal toxins, each toxin being toxic to the same insect pest, and each toxin exerting its effects through a different mode of action. In the short term this second approach is cost effective and likely sufficient to delay the development of resistance in the pest population. However, there may be at least two disadvantages to this approach as well. One disadvantage is that any development of resistance to one of the insecticidal toxins deployed into the environment of the pest immediately increases the likelihood that resistance could develop sooner than anticipated to a second or even a third toxin. Also, there is a limited number of insecticidal crystal protein toxins that are toxic to the same insect pest that are available for use that, when combined with another insecticidal protein, would fall within the defined scope of exerting its effects through a different mode of action than toxins presently in use. Therefore, additional compositions and methods for controlling pest infestation are needed, and in particular, methods and compositions are needed for use in delaying or minimizing the development of resistance to present pest control agents.
Chemical pesticidal agents typically exert their effects by inhibiting one or more proteins within the target pest either by binding irreversibly to an active site within a particular protein, by inhibiting the protein from acting upon a naturally occurring substrate, or by poisoning a respiratory or chemical gradient pathway. Recombinant methods and compositions have typically targeted cell membrane systems by producing proteins that, upon ingestion by a pest, introduce pores that result in the loss of chemical or other gradients across disrupted cell membranes. Other than chemical compositions that directly exert their effects upon proteins involved in transcription or translation mechanisms, no method has been reported for controlling pest infestation by inhibiting in the target pest the production of essential proteins through RNA mediated interference by providing one or more double stranded RNA molecules in the diet of the pest.
Antisense methods and compositions have been reported in the art and are believed to exert their effects through the synthesis of a single-stranded RNA molecule that in theory hybridizes in vivo to a substantially complementary sense strand RNA molecule. It is believed that the antisense methods function in much the same way as double stranded RNA mediated interference methods are believed to function, except that the effectiveness of the antisense response is often substantially less than desirable, intermittent, or not evident at all. Furthermore, there has never been a report in which antisense was contemplated as a means for suppressing expression of a gene in a cell remote from the cell or biological system in which the antisense sequence was expressed. Antisense technology has only been applied as a means for achieving gene-specific interference of expression within the cell or biological system in which the antisense sequence is expressed. Antisense technology has been difficult to employ in many systems for three principle reasons. First, the antisense sequence expressed in the transformed cell is unstable. Second, the instability of the antisense sequence expressed in the transformed cell concomitantly creates difficulty in delivery of the sequence to a host, cell type, or biological system remote from the transgenic cell. Third, the difficulties encountered with instability and delivery of the antisense sequence create difficulties in attempting to provide a dose within the recombinant cell expressing the antisense sequence that can effectively modulate the level of expression of the target sense nucleotide sequence.
The phenomenon of double-stranded RNA (dsRNA) induced silencing has been known for a number of years in plant systems. One form of dsRNA induced silencing is referred to as co-suppression and as virus-induced gene silencing (VIGS) and is reviewed in Matzke et al. (Adv. Genet., 2002, 46:235-275). Co-suppression and VIGS effects in recombinant plant systems were observed but unexplained before dsRNA was identified in animal systems as the trigger for induction of the evolutionarily conserved mechanism of gene suppression. Guo et al. first observed that the use of sense RNA as a control was as effective as antisense RNA in specific silencing of a targeted gene in C. elegans (Guo et al., 1995, Cell 81:611-620). Fire et al. suspected that the single stranded RNA preparations used by Guo et al. were contaminated with dsRNA, and subsequently demonstrated that dsRNA was a much more potent trigger than single stranded RNA for achieving gene specific silencing. The observations by Fire et al. distinguished the physical attribute of double stranded RNA suppression from antisense suppression (Fire et al., 1998, Nature 391:806-811). It is believed that the post-transcriptional gene silencing effects observed in plants (Jorgensen, 1990, Trends Biotechnol. 8:340-344) and the quelling effect in fungi (Romano et al., 1992, Mol. Microbiol. 6:3343-3353; Bernstein et al., 2001, RNA 7:1509-1521) using single stranded RNA is a result of contamination of samples with double stranded RNA sequences (Dykxhoorn et al., 2003, Nature Reviews 4:457-467; Hannon et al., 2002, Nature 418:244-251). It is now clear, however, that double stranded RNA mediated inhibition of gene expression, co-suppression, and virus-induced gene silencing are triggered by dsRNA and operate by similar mechanisms (Stevenson, 2003, Nature Reviews 3:851-858; Bernstein et al., 2001, RNA 7:1509-1521).
The lack of understanding of the specific mechanisms involved in these phenomena has meant that there have been few improvements in technologies for modulating the level of gene expression within a cell, tissue, or organism, and in particular, a lack of developed technologies for delaying, repressing or otherwise reducing the expression of specific genes using recombinant DNA technology. Furthermore, as a consequence of the unpredictability of these approaches, no commercially viable means for modulating the level of expression of a specific gene in a eukaryotic or prokaryotic organism is available.
Double stranded RNA mediated inhibition of specific genes in various pests has been previously demonstrated. dsRNA mediated approaches to genetic control have been tested in the fruit fly Drosophila melanogaster (Tabara et al., 1998, Science 282:430-431). Tabara et. al. describe a method for delivery of dsRNA involved generating transgenic insects that express double stranded RNA molecules or injecting dsRNA solutions into the insect body or within the egg sac prior to or during embryonic development. Research investigators have previously demonstrated that double stranded RNA mediated gene suppression can be achieved in nematodes either by feeding or by soaking the nematodes in solutions containing double stranded or small interfering RNA molecules and by injection of the dsRNA molecules. Rajagopal et. al. described failed attempts to suppress an endogenous gene in larvae of the insect pest Spodoptera litura by feeding or by soaking neonate larvae in solutions containing dsRNA specific for the target gene, but was successful in suppression after larvae were injected with dsRNA into the hemolymph of 5th instar larvae using a microapplicator (J. Biol. Chem., 2002, 277:46849-46851). Similarly, Mesa et al. (US 2003/0150017) prophetically described a preferred locus for inhibition of the lepidopteran larvae Helicoverpa armigera using dsRNA delivered to the larvae by ingestion of a plant transformed to produce the dsRNA. It is believed that it would be impractical to provide dsRNA molecules in the diet of most invertebrate pest species or to inject compositions containing dsRNA into the bodies of invertebrate pests. The diet method of providing dsRNA molecules to invertebrate pests is impractical because RNA molecules, even stabilized double stranded RNA molecules, are in effect highly unstable in mildly alkaline or acidic environments such as those found in the digestive tracts of most invertebrate pests, and easily degraded by nucleases in the environment. Therefore it is unlikely that feeding dsRNA to a lepidopteran larvae having a digestive tract pH in the extreme alkaline range would result in the suppression of any gene within the cells of the larvae. It would also be impractical to formulate compositions containing dsRNA molecules, such as recombinant bacteria expressing such dsRNA molecules, as a food source for nematodes, that could be applied to broad expanses of soil to control invertebrate pest infestations of crop plants.
Therefore, there exists a need for improved methods of modulating gene expression by repressing, delaying or otherwise reducing gene expression within a particular invertebrate pest for the purpose of controlling pest infestation or to introduce novel phenotypic traits.